The purpose of this proposal is for Dr. Zuckerman to obtain intensive training in embryonic stem (ES) cell biology. He will train in the laboratory of Dr. Martin Pera in the Institute for Reproduction and Development at Monash University in Melbourne, Australia. Dr. Pera is a pioneer in human ES cell technology and a contributer of nearly half of the human ES cell lines that are approved by NIH and are currently available. All of the training and experiments done under this grant will utilize these NIH-approved ES cell lines (ES01- ES06). Training will include technical training in isolation, identification, and maintenance of ES cells in culture, techniques for inducing ES cell differentiation into cells of desired lineages, and methods of extrinsic DNA transfer into ES cells. There will be extensive guided and self-driven reading of ES cell and related scientific and ethical literature. There will be frequent discussions with Dr. Pera, weekly lab meetings for discussion of recent results, and frequent seminars on topics relevant to ES cell biology. There is a rich stem cell research environment in Melbourne. Hematopoietic stem cell biology pioneer Don Metcalf and his extended group are at the Walter and Eliza Hall Institute, and Paul Simmons and his group at the Peter MacCallum Cancer Institute work on stem cell plasticity. Until recently, human ES cells could only be grown and maintained in the undifferentiated pluripotent state over a mouse embryo fibroblast feeder cell layer. Bone Morphogenic Protein (BMP)-2 is produced by the ES cells and/or early differentiated progeny, especially under conditions of stress. BMP-2 drives differentiation of ES cells into endodermal cells. Gremlin, a DAN family member, which is upregulated by BMP-2, has been shown recently to be a relatively specific antagonist of BMP-2 and BMP-4. In preliminary studies, Dr. Pera found that feeder cells in ES cell cultures produce gremlin transcripts. We will test the hypothesis that gremlin, by interfering with BMP-2 function, may play a large role in the ability of the feeder cells to maintain ES cells in the undifferentiated state. We will examine the ability of gremlin to prevent ES cell differentiation in cultures with no feeder cells and the ability of "impotent" feeder cells to support ES cell self-renewal and growth after being transfected with gremlin cDNA and overexpressing gremlin protein. We also will determine whether inhibition of gremlin production abolishes the ability of a functional feeder cell layer to maintain ES cells in the undifferentiated state.